间变性淋巴瘤激酶（ALK、CD247）是基于抗体的治疗的潜在靶点。然而，目前还没有针对 ALK 的抗体疗法进入临床试验，因此需要开发具有独特治疗优点的新型抗体。与全长抗体相比，单域抗体 (sdAb) 具有治疗优势，包括更深的肿瘤渗透、经济高效的生产以及从正常组织中快速清除。在这项研究中，我们从内部噬菌体库中鉴定了人免疫球蛋白重链可变结构域（VH 结构域）（VH20）。 VH20 表现出良好的可开发性和高特异性，与约 6000 种人膜蛋白没有脱靶结合。 VH20 有效结合 ALK 富含甘氨酸的区域，EC50 为 0.4 nM，KD 为 6.54 nM。基于 VH20 的双特异性 T 细胞接合器 (TCE) 和嵌合抗原受体 T 细胞 (CAR T) 都以 ALK 依赖性方式对表达 ALK 的肿瘤细胞表现出有效的细胞溶解活性。与 ALK 阳性细胞孵育后，VH20 CAR T 特异性分泌促炎细胞因子，包括 IL-2、TNFα 和 IFNγ。据我们所知，这是第一个报道的针对 ALK 的人类单域抗体。我们的体外表征数据表明，VH20 可能是一种有前途的 ALK 靶向 sdAb，在表达 ALK 的肿瘤中具有潜在应用，包括神经母细胞瘤 (NBL) 和非小细胞肺癌。

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC50 of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.